Membrane reagent addition devices have been used to improve detection in modern high performance liquid chromatography (HPLC). One example of a membrane reagent addition device is disclosed in U.S. Pat. No. 4,448,691 to The membrane reagent addition device taught by that patent utilized relatively long lengths of tubular form membranes of a relatively small pore size or relatively long lengths of nonporous tubular membranes, suspended in a space containing a reagent for permeation transfer of an effective amount of the reagent through the membrane and into the liquid chromatographic carrier or flow injection analysis carrier.
Several problems are associated with the prior membrane reagent addition devices. The relatively long tubular membranes are subject to being damaged during device construction by becoming pinched in the seams of the device and during operation can become tangled in a stirrer used to stir the reagent. In addition, the relatively long tubular membranes cause deleterious bandspreading which can reduce the resolution of closely eluting peaks in chromatographic applications and can reduce the sampling frequency in FIA applications.
The present invention solves these problems by using a short length of relatively large pore size tubular membrane (or a limited area of sheet form membrane if equivalently utilized). The relatively short length of membrane reduces bandspreading significantly to as low as 50 .mu.l or less. In addition, a short length of membrane is less likely to become pinched in the seams of a membrane reagent addition device during device construction and can be protected from damage from a stirring bar by covering the membrane with a high strength perforate structure.
The prior art did not suggest the use of relatively large pore size membranes or the use of short lengths of tubular membranes. The relatively long lengths of tubular membranes used in the prior art were needed to permeate enough reagent into the analytical stream to be effective. Large pore size membranes were probably not used in the prior art due to the problem of excessive leakage across the membrane as discussed in detail below.
A characteristic of membrane reagent addition devices is the tendency for pressure to be higher on the carrier side of the membrane than on the reagent side. This pressure differential is caused by the structural features of the known devices and often exacerbated by the often used reaction coil or packed bed in fluid communication with the reagent addition device and the detector. The reaction coil or in the alternative, the packed bed, serves to homogenize the constituents of the carrier and in some cases, increase the time necessary to complete a reaction between the carrier components. In addition, the relatively long length of tubular membrane as used in prior membrane reagent addition devices, has an associated carrier pressure drop along the inside (bore) of the tubular membrane. Therefore, pressurizing the reagent on the outside of a relatively long tubular membrane is not an effective solution to minimize excessive leakage of carrier across the membrane into the reagent when the membrane has relatively large pores. If the reagent is pressurized to a pressure equal to theccarrier pressure in the end portion of such a tubular membrane, then excessive carrier leakage across the membrane can occur at the beginning portion of the tubular membrane. If the reagent is pressurized to a pressure equal to the carrier pressure in the beginning portion of such a tubular membrane, then excessive amounts of reagent can pass across the membrane at the end portions of the tubular membrane. If the reagent is pressurized to a pressure equal to the carrier pressure in the middle portion of such a tubular membrane, then excessive amounts of carrier can still pass across the membrane at the beginning portion of the tubular membrane and excessive amounts of reagent can pass across the membrane at the end portions of the tubular membranes.
Relatively large pore size membranes are desirable in membrane reagent addition devices despite these problems. In the prior membrane reagent addition devices the membrane often had to be selected for a specific reagent. Each reagent/membrane combination needed to be tested to ensure that enough reagent would permeate the membrane to be effective. The use of a relatively large pore size membrane offeredthe promise of an unlimited ability to permeate almost any reagent but the problems described in the previous paragraph are believed to have frustrated their use.
The present invention solves the aforementioned problem of excessive leakage across a relatively large pore size membrane by using a short length of such a tubular membrane which significantly reduces the pressure drop of carrier along the inside of the tubular membrane. Surprisingly, the short length of such a tubular membrane (or limited area of sheet form membrane if equivalently utilized) still permeates an effective amount of reagent into the carrier stream. In many applications it is still necessary to pressurize the reagent chamber with, for example, compressed air to prevent excessive carrier leakage across the membrane and pressurizing the reagent space of a membrane reagent addition device employing a short length of tubular membrane of relatively large pore size can overcome the aforementioned leakage problems. The present invention also comprises a novel means of pressurizing the reagent space of a porous membrane reagent addition device. This means is simply to hermetically seal the reagent space which has been essentially completely filled with reagent. In this event the pressure on each side of the porous membrane naturally and automatically equilibrates. Thus, whatever the carrier pressure is on the carrier side of the membrane (as a result of detector back pressure or back pressure through a mixing coil or mixing column) the reagent pressure automatically adjusts to approximately the same pressure as the carrier. Therefore, even when this embodiment of the invention is used to add reagent to a liquid chromatographic eluent stream before the eluent stream passes through the chromatographic column (and where the eluent pressure could be 2,000 psi, for example) the reagent will automatically self-pressurize and then effectively add reagent to the eluent stream.